❑ What is Urine Analysis

▪ Urine analysis also called urinalysis examination.

▪ Also known as urine R&M (routine & microscopy)

❑ Why urinalysis ?
▪ General evaluation of health.

▪ Diagnosis of disease or disorders of the kidneys or urinary tract.

▪ Diagnosis of other systemic disease that after kidney function.

▪ Monitoring of patients with diabetes.

▪ Diagnosis of jaundice and other infection.

 ❑ Urine
▪ Urine is a straw colored liquid excretory material of body which is
secreted by kidney and excreted by urethra.

▪ Is an ultrafiltrate of plasma from which glucose , amino

acids , water and other substances essential to body
metabolism have been reabsorbed .

▪ Urine carries water products and excess water out of the

body .
 Urine consists of :

(96%) (4%)
Water Dissolved solids

(2%) (2%)
Urea: (half) Other compounds

Inorganic: Organic:
CI , Na , K Creatinine
tract Uric acid
amount of
sulfate² HCO3
etc
 Urine Sample
Sample type Sampling Purpose

Random specimen No specific time most common , Routine screening , chemical

taken anytime of day
Morning Sample First urine in the morning , most Pregnancy test, microscopic
concentrated test
Clean catch midstream Discard first few ml, collect the rest Culture

24 hours All the urine passed during the day Used for quantitative and
and night and next day 1st sample is qualitative analysis of
collected substances

Postprandial 2 hours after meal Determine glucose in diabetic

monitoring
Preservation of urine sample
▪ Test to be done within 2 hrs.

▪ Can be kept at 4 -6ᵒ C for maximum 8 hr.

▪ Routine analysis – preservatives avoided.

▪ Preservative used in 24 hr. sample.

• Hydrochloric acid – adrenaline , non adrenaline , steroids

• Toluene – measurement of chemicals.
• Boric acid – general preservative.
• Thymol – inhibit bacteria and fungi.
• Formalin – formed elements.
 Urinalysis

physical Microscopic
Examination Examination
1. Volume 1. Cells
2. Color Biochemical 2. Crystals
3. Odor Examination 3. Casts
4. Turbidity 4. Microorganism
5. Rection (pH) 1. proteins 5. parasites
6. Specific gravity 2. Sugars 6. Contamination
3. Ketone bodies
4. Bile salts
5. Bile pigments
6. Blood
 PHYSICAL EXAMINATION
❑ Volume
▪ Average 24-hr urinary output in adults is 800 – 2000 ml.
▪ The volume varies according to fluid intake , diet, and climate.

▪ Abnormalities of urinary are as follows.

o Polyuria - means urinary volume > 2000 ml /24 hours.
▪ Cause :-- Diabetes mellitus
Diabetes insipidus
Chronic renal failure
Diuretic therapy
Increase water ingetion

o Oliguria - means urinary volume < 400 ml / 24 hours.

▪ Cause :-- Febrile states
Acute glomerulonephritis
Congestive cardiac failure or dehydration
o Anuria- means urinary output < 100ml / 24 hours
o or complete absence of urine output

Couse :-- Acute tubular necrosis

Hemolytic transfusion reaction
Acute glomerulonephritis
Complete urinary tract obstruction

.
Nocturia –
Increase frequency of urination in night.
 ➢ Color
▪ Normal Color – Pale yellow or amber due to the presence of pigment called
urochrome, urobilin , uroerythrin.
Abnormal color of urine:-

▪ Colorless -
Dilute urine
Diabetes mellitus ,
diabetes insipidus ,
overhydration
Red color :--
Hematuria [Presence of RBC]
Hemoglobinuria [Presence of Hb]
porphyria

Black color : -
Alkaptonuria
muscle injury
Melanin , myoglobin,
[black color due to phenol poisning or
hemoglobin convert to methemoglobin]
Yellow :-- Concentrated urine due to dehydration,
fever ,lever disease

Dark yellow – due to presence of bilirubin (jaundice)

Dark Brown – due to liver and kidney disorders

Orange colour:--
excess amount of Urobilinogen ,
porphobilinogen,
anti inflammatory drug.

Milky white :--

Chyluria , crystals , fats , UTI .
❑ Appearance
• Normal - freshly voided urine is clear transparent in appearance.

• Foamy urine – occurs in the presence of excess proteins.

• Turbid urine - Presence of abnormal number of leukocytes ,epithelial cells, bacteria.

• cloudy urine - concentrated urine ,UTI, phosphates ,uric acid , amorphous.

❑ Odor (smell)
• Freshly voided urine has a aromatic odor due to volatile organic acids.

• After standing urine develops ammoniacal odor ( formation od ammonia occurs

when urea is decomposed by bacteria).

• Some abnormal odors with associated conditions are.

Fruity :-- Ketoacidosis , starvation ,Diabetes.

Mousy or musty :-- Phenylketonuria

Fishy :-- Urinary tract infection with proteus

Ammoniacal :-- Urinary tract infection with Escherichia coli old standing urine.
❑ Reaction and pH :--
• PH is the scale for measuring acidity or alkalinity.

• Fresh urine is required for correct estimation of pH – upon standing urine

• Normal pH – 4 .7 – 7.8
Clinical significance

Acidic :-- low urine PH

Respiratory acidosis ,
UTI by e. coli , diabetes, Diarrhea,.

Alkaline :-- higher urine PH

kidney failure, diet ,
Proteus and pseudomonas infection
if urine is kept in the bladder (hold urine)
 ❑ Specific Gravity
• Specific gravity measure the concentration of solute in urine sample.
• Measure of concentrating ability of kidneys and is determined to get information about this
tubular function.

• Normal SG of urine is 1.016 to 1.022 in 24 hours sample and also depends on the state of
hydration.

• SG increases as solute concentration increases.

• SG Decrease as solute concentration decrease.

More solutes SG

Less solutes SG
Procedure –
Detected by urinometer urine is taken in to the tube in 3/4 amount.

Urinometer is introduce into the tube without touching the walls in a rotational motion.

Reading is noted.
Isosthenuria
fixed SG at 1.010 due to loss of concentrating ability of tubules seen in end stage renal failure.

Hypersthenuria :-- increased SG 1.030

Cause :- Diabetes mellitus
Fever
Dehydration

Hyposthenuria :-- decreased SG 1.003

Cause :- Diabetes insipidus
Alcohol
dilute urine
 CHEMICAL EXAMINATION
▪ The chemical examination is carried out for the following substances.

❑ Protein.

❑ Glucose.

❑ Ketones Bodies.

❑ Bilirubin.

❑ Bile Salts.

❑ Urobilinogen.

❑ Blood.
 ❖ Protein
▪ Normally kidney excrete few amount of low molecular wight
protein in urine <150mg 124 hr.

▪ After filtration most of protein is reabsorbed in the tubules.

▪ This amount of protein in urine can’t be detected by routine

test.

Cause's :-
❑ Glomerular proteinuria
▪ Due to increased permeability of glomerular capillary wall.
Ex:- Nephrotic syndrome
❑ Tubular Proteinuria

▪ Due to renal tubules dysfunction.

❑ Overflow proteinuria- low molecular weight protein

▪ Bence jones protein , hemoglobin, myoglobin.

❑ Occasionally in high fever , hypertension , heavy exercise .

Test for proteinuria

o Heat and acetic acid test.

o Sulfosalicylic acid test.

o Heller’s test.

o Reagent strip.
 Heat and acetic acid test
❖ Principle
• Proteins are denatured and coagulated upon heating to give white cloud
precipitate after addition of 3%acetic acid.

Reagent :- 3% glacial acetic acid.

❖ Method
• Fill 2/ 3rd with urine.

• Boil upper portion for 2 minutes

• If precipitation or turbidity appears after add a few drops of

3% acetic acid.
 presence of
cloudiness

Absence of
cloudiness

take5-10 Boil the upper Add 2-3 drop of

ml urine portion acetic acid for
check cloudiness
❖ Interpretation
• If turbidity or precipitation disappears on addition of acetic acid it is due to
phosphates. (Bence jounce protein).

• If turbidity or precipitation persists after addition of acetic then is due to

proteins.

• The test is semiquantitative and can be graded from traces to 4 + depending

upon amount of protein.

▪ No cloudiness :- Negative

▪ Faint cloudiness :- Traces (less than 0.1 g/dl).

▪ Cloudiness without granularity :- + (0.1 g/dl).

▪ Granular cloudiness :- ++ (0.1- 0.2 g/dl).

▪ Precipitation and flocculation :- +++ (0.2 – 0.4 g/dl).

▪ Thick solid precipitation :- ++++ (0.5 g/dl).

 Sulfosalicylic Acid Test
❖ Principle
• Addition of sulfosalicylic acid to the urine causes formation of a white
precipitate if protein are present.

Reagent :- Acetic acid ,sulfosalicylic acid.

❖Method
• Take 2ml of clear urine in a test tube.

• Add 2-3 drops of sulfosalicylic acid (3to5%) and examine for turbidity
against a dark background.
 Negative 1+ 2+ 3+ 4+

❖ Interpretation
• Appearance of turbidity which persists after heating indicates.
 Heller’s Test
Reagent :- Nitric acid.

❖ Method
• Take 2ml of concentrated nitric acid in a test tube.

• Add urine drop by drop by the side of test tube.

❖ Interpretation
• Appearance of white ring at the junction indicates
presence of protein.
 Heller’s Heller’s
negative positive

White ring White ring

absent present

Protein Protein
absent present

HELLER’S TEST
 Reagent strip method
❖ Principle
• The reagent area of the strip is coated with bromophenol blue indicator and
buffered to an acid pH which changes color in the presence of proteins.

❖ Method
• Bromophenol coated strip is dipped in urine.

• Change in color of strip indicates presence of proteins in urine and is

compared with the color chart provided for semiquantitative grading.
 ❖ Glucose
• Glucose in urine is detection of unsuspected diabetes mellitus.

• All of the glucose filtered by the glomeruli is reabsorbed by the proximal renal tubules and
returnees to circulation.

• Normally a very small amount of glucose is excreted in urine that cannot be detected by the routine
tests.
• Presence of detectable amount of glucose in urine is glycosuria.

❖ Causes of glycosuria
1.Glycosuria with hyperglycemia
a) Diabetes mellitus
b) cushing’s syndrome.
c) Hyperthyroidism , pancreatic disease.

2. Central nervous system Diseases.

3. Liver disorders.
 Test for glucose
❖ Benedict’s qualitative test
▪ Benedict’s qualitative reagent (Blue color)
Copper sulphate :- 17.3 gm
Sodium carbonate :- 100 gm
Sodium citrate :- 173 gm
Distilled water :- 1000 ml

❖ Principle
• When urine is boiled in benedict’s qualitative solution blue alkaline
copper sulphate is reduced to red brown cuprous oxide if reducing
(glucose) agent is present.

• The color of cuprous oxide formed depend on the amount of reducing

substance present in the urine and help in semi quantitative
measurement.
❖ Method
• Add 5 ml of reagent.

• Add 0.5 ml (8 drops) of urine mix well.

• Boil over a flame for 2 minutes.
• Allow to cool at room temperature.
• Note the color change if any.

Add 5ml Add drop Note color if

reagent of urine any change
and boil
❖ Interpretation
• The result is reported in grades as follows.

No
Reducing Green Green +yellow Yellow orange Red
sugar Trace + ++ +++ ++++
 Reagent strip method
• This test is specific for glucose.

• It is based on glucose oxidase – peroxidase.

• Reagent area of the strips is impregnated with two enzymes – glucose oxidase
and peroxidase and a chromogen.

Glucose + oxygen glucose Glucose acid + hydrogen peroxide.

Oxidase
Hydrogen peroxide + chromogen peroxidase oxidized chromogen+ H2o .

• Nature of chromogen and buffer system differ in different strips.

• The strip is dipped into the urine sample and color is observed after a specified
time and compared with the color chart provided.
 CHEMICAL EXAMINATION
▪ The chemical examination is carried out for the following substances.

❑ Protein.

❑ Glucose.

❑ Ketones Bodies.

❑ Bilirubin.

❑ Bile Salts.

❑ Urobilinogen.

❑ Blood.
 Ketone bodies

(Ketone in urine indicate uncontrolled diabetes)

▪ When there is not enough insulin our body is

unable to use sugar for energy.

▪ When the body start to burn fat for energy a

group of molecule ketones are produced.
▪ There types of ketone bodies include:-
i. Acetoacetic acid
ii. beta Hydroxybutyrate
iii. Acetone
[This combination make your blood to acetic which can change the normal
function of internal organ like – liver , kidney , brain]

❖ Important terms
o Ketonuria - high amount of ketone bodies in urine.

o Ketonemia - high con of ketone bodies in blood.

o Ketoacidosis – serious diabetes complication where the body produce

excess amount of ketones (blood acid).

▪ It is life threatening condition resulting (high level of

ketone & sugar) diabetic ketoacidosis , diabetic acidosis.

▪ Normal plasma level of ketone bodies - <1mg/ dl.

▪ Normal urinary level of ketone bodies - <3mg / dl

❖ Cause's
• Diabetes mellitus.
• Starvation.
• Pregnancy.
• Excessive vomiting.
• Fasting.

❖ Test for ketone bodies

▪ Ketone bodies are usually absent in urine.

(Test) (Use)
Rothera's test For acetone ,Acetoacetic acid
Reagent strip test For acetone ,Acetoacetic acid
Hart’s test Beta hydroxy butyric acid
Gerhardt’s test (ferric chloride) Acetoacetic acid
 Rothera’s Test
❖ Principle
▪ Acetoacetic acid or acetone reacts with nitroprusside in alkaline
solution of form a purple – colored complex or purple color ring.

❖ Reagent
• Ammonium sulphate

• Sodium nitroprusside

• Liquor ammonia(ammonium hydroxide)

❖ Method
• Take 5ml of urine in a test tube and saturate it with ammonium
sulphate.
• Add a small crystal of sodium nitroprusside mix well.
• Slowly run along the side of the test tube liquor ammonia to form a
layer.
• Immediate formation of a purple colored ring at the junction of the
tube indicates a positive test.

Rothera’s powder
purple colored ring
Ammonium hydroxide

5ml of urine Rothera’s positive

 ❖ Reagent strips for ketone bodies
• It can detect as little as 5mg /dl of acetoacetic acid in urine.

• Acetoacetic acid +sodium nitroprusside Alkaline PH purple or magenta color

complex.

• Beta hydroxybutyric acid is not detected by reagent strip test.

 Bilirubin
• Bilirubin is the yellow pigment.

• Bilirubin is the end product of RBC breakdown.

• Increased formation of bilirubin cause jaundice.

causes:-
Liver disease
Liver cirrhosis
Hepatitis
Bile duct obstruction
 RBC
Red blood cell After 120 days
Hemoglobin

Heme Globin (protein - amino acid)

RE system Irone Porphyrin
Bilirubin Unconjugated bilirubin
UCB + albumin ]- blood circulation
UCB + glucuronic acid [by UGT enzyme]
Liver
Conjugated bilirubin
CB enter into gall bladder
small intestine [CB]
 Intestine (CB)

CB Urobilinogen
by micros

Urobilinogen in
circulation
Stercobilinogen

Kidney Excrete
Urobilinogen through stool
convert into urobilin Excrete
through urine
 Fouchet's Test
❖ Principle
▪ Barium chloride react with sulphate in urine form barium sulphate.
If bilirubin present in urine it adhere to barium sulphate and precipitate then its
react with fouchet reagent and oxidized bilirubin into(Green) color complex

yellow bilirubin Fouchet's reagent green bilirubin

Oxidize
❖ Reagent
▪ Ferric chloride
Fouchet's reagent
▪ Trichloroacetic acid

▪ Barium chloride
❖ Procedure
• Take 5ml of fresh urine in test tube.
• Add 2.5ml of 10% of barium chloride mix well.
• Filter to obtain the precipitate on a filter paper.
• To the precipitate on the filter paper add one drop of fouchet's reagents.
• Immediate development to blue green color around the drop indicates
presence of bile pigments or bilirubin in urine.

Add

Fouchet's reagent

precipitate on blue green

a filter paper. color
Reagent strips for bilirubin or bile pigments:-

Bilirubin + 2,4 Dichloroaniline acidic medium azobilirubin

(pinkish tan color)
 Urobilinogen
• Urobilinogen is a colorless compound.
• Urobilinogen is a bilirubin degradation produce that is
formed by the action of intestinal bacteria on conjugated
bilirubin.
• Urobilinogen is normally found in the urine but in
trace amount.

• Normal value :- 0.2 – 1.0 mg/dl

• High urobilinogen :- > 1.0mg/dl
• Low urobilinogen :- < 0.2 mg dl

Stercobilinogen -- yellow – brown color of stool

Urobilin -- yellow color of urine

 Test for Urobilinogen
I. Ehrlich aldehyde test
II. Reagent strip test

I. Ehrlich aldehyde test

❖ Principle
• Ehrlich reagent para amino benzaldehyde in concentrated HCI
reacts with urobilinogen to form aldehyde which is red color
complex
❖ Reagent
Ehrlich Reagent
a. Concentrated HCI acid -100ml.
b. Dimethylamine benzaldehyde - 4gm.
c. Distilled water - 400ml

❖ procedure
▪ Take 5ml fresh urine in a test tube .

▪ Add 0.5ml Ehrlich reagent.

▪ Allow to stand for 5 mint at R.T.

▪ Observe result.
5ml Add 0.5ml Pink (normal urobilinogen)
urine Ehrlich
reagent
Dark red (high urobilinogen)

Pink color - normal amount of urobilinogen

Dark red color - increased amount of urobilinogen

 II. Reagent strip test
P dimethylamine benzaldehyde + urobilinogen Acidic medium Pink color
( Indicator )
 Bile salts
• Bile salts are primary component of bile it help in excretion
of bile pigment (bilirubin) through bile .

• Help in metabolism of fat and vitamin (fat soluble).

• Bile salt is reabsorbed in the ileum and return to the liver

through the portal circulation for Re-secretion.

• Normally bile salt are not present in urine.

• Bile salts are detect in urine in various condition.

i. Obstructive jaundice
ii. Cirrhosis Liver disease (jaundice)
iii. Hepatis
 Test for bile salts
Hay’s test (hay’s Sulphur Powder test)

❖ Principle
▪ Bile salts present in urine reduce the surface tension of urine resulting in the
shrinking of finally Sulphur powder when layer on the surface of urine.
❖ procedure
• Sulfur powder is sprinkled over the surface of urine.

• If bile salt are present – sulfur sink on bottom.

• If bile salt are absent – sulfur float on the surface.

Sulfur
granules
Sulfur
granules
Water as Bile salt
control positive
 Blood
• Blood may be found in urine in the form of RBC or when
hemolyzed in the form of hemoglobin.

• Hematuria -- presence of RBC in urine.

• Hemoglobinuria – presence of hemoglobin in urine.

• Cause – due to hemolytic transfusion reaction.

Some infection
RBC hemolysis
 Benzidine Test
❖ Principle
• In the presence of Hydrogen peroxide (H2 O2) Benzidine solution
and the liberated active oxygen , this oxidize organic compound benzidine
form green or blue color complex if the hemoglobin or RBC present.
❖ Reagent
• Benzidine reagent
• Hydrogen peroxide (H2 O2)
❖ Procedure
• Add 3 ml of benzidine in a test tube.

• Add 2ml of H2 O2 and mix well.

• Then add 3ml of urine wait for 3-5 mint.

• Observe color change.

❖ Result
Fait green Trace
Green +
Bluish green ++
Blue +++
Deep blue ++++
 Dipstick /reagent strip method.

Detect both HB (hemoglobinuria) and RBC (Hematuria)

Acidic medium
Tetramethyl benzidine + H202 + Hb blue or green color complex.
MICROSCOPIC EXAMINATION OF URINE
❑ Introduction
▪ The microscopic examination is a valuable diagnostic tool for the detection
and evolution of renal and urinary tract disorder and other systemic disease.

Diagnosis of :-
• Kidney stones
• Tumors
• Traumatic injury of urinary tract
• UTI
• Nephritis
 Crystal WhatsApp Nu. - 9977002457
Elements which are detect in urine :-
Cell Cast Normal urine

RBC
Cellular Non Cellular Acetic Alkaline
WBC • Amorphous phosphates
Epithelial • Red cell • Granular cast • Calcium oxalate
• Calcium carbonate
cell cast • Hyaline cast • Uric acid crystals
• White cell • Waxy cast • Amorphous • Calcium phosphates
cast urates • Ammonium biuret
• Epithelial • Hippuric acid • Triple phosphates
cell cast crystals
Abnormal urine crystal
Other
• Cystine
Bacteria • Leucine
parasite • Tyrosine
yeast cell • Cholesterol
Spermatozoa • bilirubin
❑ Principle
▪ The microscopic element present in urine (suspension) are collected in the
form of deposit by centrifugation.
▪ A small drop of sediment is examined by making a coverline preparation
under microscopic.

❑ Requirement
• Centrifuge
• Test tube (10x 75 mm)
• Glass slide
• Cover slip
• Microscope
• Pasteur pipettes
 WhatsApp Nu. - 9977002457

❑ Specimen
• Freshly voided
• Midstream
• Morning urine (most concentrated)

❑ Procedure
▪ The urine contain soluble and solid particle in suspension .
These solid particle are separated out from suspension by
centrifugation and this is known as sediment.

• About 10ml urine take in test tube.

• Centrifuge for 5 minutes at 2,500 rpm.

• Supernatant (fluid above the sediment) is carefully discarded.

▪ Resuspend the deposit by shaking the tube.

▪ Place one drop of the deposit on a glass slide.

▪ Cover it with cover slip.

▪ Observe under low power objective and urine sediment can be

observe unstained or using commercial stain.
 Red Blood Cells
o Few (0-2 / HPF)

• RBC is not present normally in urine.

• Increased RBC known as hematuria .

Couse :- Urinary system disease trauma.

• In fresh urine these cells have a normal, pale or yellow appearance.

• Biconcave shape about 7um diameter , 2um thick.

• In dilute (hypotonic) urine red cell swell up and lyse.

• In concentrated (hypertonic) urine red cell crenate.

Note :- Yeast cell can be mistaken for RBCs so avoid yeast cell.
WhatsApp Nu. - 9977002457
 WBC or PUS Cells
o 2-3 Pus cell / HPF

• These are mostly neutrophils 10-12 µm.

• Identify by nucleated structure (dark dot inside the cell).

• Increased number of WBC in urine called pyuria.

Couse :- Indicate renal infection

bladder (cystitis)
WhatsApp Nu. - 9977002457
 Epithelial Cells
o 3-5 / HPF

• These cell may originate from any site in the genitourinary tract
from the proximal convoluted tubule to the urethra or vagina.

• Can be found due sloughing off old cells .

• 3 main types of epithelial cell.

WhatsApp Nu. - 9977002457
 EC

Squamous EC Transitional EC Tubular EC

• Contain small • Pear shaped or round • Cuboidal, flat or columnar
nucleus flat and • Contain two nucleus • Contain large round nucleus
irregular and large • Cell carcinoma • Tubular necrosis
(vaginal fluid) (cancer of urinary tract) • Pyelonephritis
(bladder)
 WhatsApp Nu. - 9977002457

Cast

▪ Urinary East are formed in the lumen of the tubules of the kidney.

▪ Renal tubules secrete small amount of mucoprotein (Tamm – Horsfall

protein).

▪ In contain of slow urine flow, acidic PH, and increased solute this protein
accumulates and begins to gel forming cast.

▪ Cast formation takes place in the distal and collection tubules.

 Cast

Cellular Non Cellular

• Red cell cast • Granular cast
• White cell cast • Hyaline cast
• Epithelial cell cast • Waxy cast
 WhatsApp Nu. - 9977002457

Noncellular Cast
Colorless, rounded end (like Pathologic seen in
Hyaline cast glass) homogeneous glomeruli tubular
disease

Covered with granules Glomerular disease

Granular cast

Yellow grey or colorless high Renal disease

Waxy cast refractive index amyloidal disease
Hyaline cast granular cast
Waxy cast
 WhatsApp Nu. - 9977002457

Cellular Cast
glomerulonephritis
Red cell cast

pyelonephritis
White cell cast
Glomerular disease

tubular necrosis
Epithelial cell cast Chronic renal
disease
 ❑ Micro organism
o Not present in urine

Bacteria :-
• Tiny round or rod shaped.

• Contamination may occur from bacteria present in urethra , vagina or

other external source.

• Gram- Negative bacilli - E.coli , pseudomonal , proteus.

Gram - Positive cocci - enteron cocci , streptococcus pyogenes.
 WhatsApp Nu. - 9977002457

Yeast cells :
• Smooth colorless and ovoid and often seen budding or in hair.

• Yeast cell are often mistaken for RBC.

• For differentiating BLW yeast and RBC add one drop of the acetic acid to a
drop of urine sediment.

• Found in UTI , diabetic patient, skin or vaginal contamination.

Parasite

commonly parasite in urine are:-

Trichomonas vaginalis
filaria
 WhatsApp Nu. - 9977002457

Crystals
Many of the crystals that are found in the urine have little clinical significance although they
may be found in calculus formation , metabolic disorders and in the regulation of medication.
Crystals

Normal urine Ab normal urine

• Cystine
• Leucine
Acetic Alkaline • Tyrosine
• Calcium oxalate • Amorphous phosphates • Cholesterol
• Uric acid crystals • Calcium carbonate • Sulphur crystals
• Amorphous urates • Calcium phosphates
• Hippuric acid crystals • Ammonium biuret
• Triple phosphates
Normal urine crystal (acidic)
Elements Appearance Pathology and non pathology

Calcium oxalate Colorless and Present after ingestion of

envelope tomatoes , spinach ,garlic ,
shaped vitamins
(dumbbell They can be present in increased
shape) nu. In diabetes mellitus liver
disease.
uric acid crystal diamond Gout , chronic nephritis , acute
rhombic or febrile condition
rosette yellow
or red brown.
Elements Appearance Pathology and non WhatsApp Nu. - 9977002457
pathology
Amorphous urates Yellow –red granular No clinical
significance
(sodium potassium ,
magnesium and
calcium)

Hippuric acid Elongated prisms or plates No clinical

significance
Normal urine crystal (alkali)
Elements Appearance Pathology and non pathology

Calcium Small colorless , No clinical significance

carbonate granular and
spherical

Calcium Long thin No clinical significance

phosphates colorless (as
needle)
 WhatsApp Nu. - 9977002457

Elements Appearance Pathology and

non pathology
Ammonium biurets Spherical bodies , If found in fresh
yellow brown urine they are ab
normal

Triple phosphates Colorless prism with Chronic cystitis ,

three to six side. chronic pyelitis
enlarged prostate
 WhatsApp Nu. - 9977002457
Abnormal crystal urine

Cystine :- Seen in amino acid metabolism.

Turn red in the presence of sodium nitroprusside.

Leucine :-. Liver damage.

Tyrosine :-. Needles arranged in sheaves.

Liver disease.

Cholesterol:-. Regular irregular transport.

Seen in renal disease.

bilirubin:- Liver disease

Cholesterol cystine
Tyrosine

Leucine bilirubin
 CEREBROSPINAL FLUID (CSF)
❑ The cerebrospinal fluid (CSF) is a clear, colorless transparent fluid present in the
cerebral ventricles spinal canal and subarachnoid spaces.

CSF formation
▪ CSF is largely formed by the choroid plexus of the lateral
ventricle and remainder in the third and fourth ventricles.

▪ CSF is a selective ultrafiltrate of plasma.

▪ Volume -150ml.
Diagnosis of
1. Meningitis( bacteria ,virus , protozoa)

2. Infections disease of brain and spinal cord

3. Inflammation.

4. Spinal cord tumor.

Nature :- Characteristics of CSF WhatsApp Nu. - 9977002457

Color - Colorless
Specific gravity - 1.004 -1.007
Reaction (PH) - (7.3 - 7.4)
Appearance - Clear, transparent

Total solids :-
Protein - 15 to 45mg /dl (Albumin = 50 – 70%) ,(Globulins = 30 – 50%)
Glucose - 40 – 80 mg/dl
Chlorides - 700 – 750 mg/dl
Sodium - 144 – 154 mEq/l
Potassium - 2.0 – 3.5 mEq/l
Creatinine - 0.5 – 1.2 mg/dl
Cholesterol - 0.2 – 0.6 mg/dl
Urea - 6 – 16 g/dl
Uric acid - 0.5 – 4.5 mg/dl

Cells :- 0.8 Lymphocytes/ per cu mm (ul). (Neutrophils :- Absent)

Function
▪ Provide fluid cushion for protection and support the brain the spinal cord. (by
act like a fluid buffer).

▪ Act as shock absorber for brain and spinal cord.

▪ Carries nutrient and remove waste product.

▪ Maintain a constant pressure inside the brain and around the spinal cord.

▪ It keep moist (brain and spinal cord).

▪ It act as a medium for the transfer of substance form tissue of the brain spinal
cord to the blood stream.
WhatsApp Nu. - 9977002457
Collection WhatsApp Nu. - 9977002457
▪ Cerebrospinal fluid is collected by lumbar puncture (LP) under strict
aseptic conditions similar to any surgical procedure.
▪ An experienced physician or a specially trained nurse dose the LP and
specimen collection.
▪ After properly positioning the patient the sterile LP needle with the
stylet is inserted between the[L3and L4] Third and fourth lumbar
vertebrae to a depth of 4-5cm.

▪ The stylet is withdrawn and the fluid flows freely into a sterile test tube
through the needle.

▪ A total amount between 6 - 7 ml of CSF is collected in 3 sterile tubes,

which are chemically cleaned and washed with distilled or deionized
water.
▪ The needle is remove the area is cleaned and bandage over
the needle site.
 Tube 1 Tube 2 Tube 3

1ml 2.5ml 2.5ml

Bacterial Chemical Microscopic
examination examination examination

After collection test start within hr.

Do not refrigerate sample if bacteriological examination

 WhatsApp Nu. - 9977002457

Physical examination

Chemical examination.

Microscopic examination.
 CSF EXAMINATION
❑ Tube 1 – Used for bacterial culture.

❑ Tube 2 – For biochemical test.

❑ Tube 3 – Centrifuge / Non centrifuge.

Sediment

(a) Prepare 3 smear.

o DLC
o Gram stain
o ZN stain

(b) Wet mount for Trypanosoma.

(c) India ink preparation.
 WhatsApp Nu. - 9977002457

CSF Examination

Physical ex. Microscopic Ex. Biochemical Ex.

Cell counting Microbiological

TLC DLC Z-N stain Wet mount Culturing

Gram stain India ink

preparation
❖ Physical Examination
❑ Observe the specimen and not down.
1. Color – Colorless.
• Red color – Presence of blood.
• Yellow color – Presence of bilirubin (known as xanthochromia).

2. Appearance – clear.
• Cloudy – Due to pus formation.

3. Specific Gravity --
• Determined by refractometer.

4. PH --
5. Clot formation -- Normal CSF does not clot.
• Clot formation is the presence of fibrinogen – fibrin to produce clot
(when BBB is disturbed)
Nature :- Characteristics of CSF WhatsApp Nu. - 9977002457

Color - Colorless
Specific gravity - 1.004 -1.007
Reaction (PH) - (7.3 - 7.4)
Appearance - Clear, transparent

Total solids :-
Protein - 15 to 45mg /dl (Albumin = 50 – 70%) ,(Globulins = 30 – 50%)
Glucose - 40 – 80 mg/dl
Chlorides - 700 – 750 mg/dl
Sodium - 144 – 154 mEq/l
Potassium - 2.0 – 3.5 mEq/l
Creatinine - 0.5 – 1.2 mg/dl
Cholesterol - 0.2 – 0.6 mg/dl
Urea - 6 – 16 g/dl
Uric acid - 0.5 – 4.5 mg/dl

Cells :- 0.8 Lymphocytes/ per cu mm (ul). (Neutrophils :- Absent)

 WhatsApp Nu. - 9977002457

❖ Microscopic Examination

Cell counting Microbiological

TLC DLC Z-N stain Wet mount Culturing

Gram stain India ink

preparation
 Total Leukocyte Count
▪ Lymphocyte present normally in few amount about 0-5 WBC//cu mm
Increased cell count in CSF is known as pleocytosis.

o Specimen – Uncentrifuged specimen if 3 tube

2 Tube (sediment use)

o Equipment – Microscope
Cover slip
Graduated pipette
Fuchs - Rosenthal counting chamber
or Neubauer chamber
Fuchs - Rosenthal counting chamber
o CSF Diluting Fluid –
• 2% Glacial acetic acid – 0.3g
• 3g/dl Methylene blue – 10 drops
• Distilled water – 100ml
❖ Procedure
▪ Mix the CSF sample carefully .
▪ Make 1:20 dilution
▪ Dilute the specimen only when it cloudy. If clear CSF
use it undiluted.

by using WBC pipette.

 WhatsApp Nu. - 9977002457
If use pipette – 20ml CSF diluting fluid.
1ml specimen in a test tube.

▪ Wait for 5 mint after diluting the specimen.

▪ Load the counting chamber.

▪ Leave the chamber on the counter for 5 mint to allow the cell to settle.

▪ Count under low power objective (10x) which cover

Count all the 16 squares
Calculation = Number of cell counted x dilution
Area counted x depth of fluid

N x 20 N X 10 X 20
=
9 x 0.1 9

Increased Leucocyte – Infective meningitis.

 DLC(differential Leukocyte count)
o Information regarding the relative distribution of various
WBC – Lymphocyte , Neutrophils , and Other.

❑ Specimen – Make thin smear.

❑ Equipment
• Microscope
• Centrifuge
• Pipette
• Romanowsky stain
• Slide
 WhatsApp Nu. - 9977002457
❖ Procedure
▪ Centrifuge the specimen.
▪ Mix the deposit and take a drop of the sediment on a clear slide and make smear.
▪ Leave it to dry and fix with methanol and stain with Romanowsky (Leishman stain).

▪ Examine cell under the microscope using 40 x objective.

❖ Result
▪ Normal CSF contain few lymphocytes.

▪ High count – Pyogenic infections.

▪ High lymphocyte count – mycotic infection , TB , viral

meningitis , trypanosomiasis.
 Wet Mount Of CSF
o Performed in case of sleeping sickness (trypanosomiasis and
cryptococcosis).
 WhatsApp Nu. - 9977002457
India Ink Preparation

o For cryptococcal infection.

❑ Specimen – CSF Sediment.

❑ Result – Round budding spores (Surrounded by colorless capsule)

MICROBIOLOGICAL EXAMINATION
Microbiological examination

Gram stain culturing

Z-N stain
Wet mount
India ink
preparation
❑ Gram stain
Reaction –

Morphology –

organism that commonly cause meningitis are –

▪ Neisseria meningitidis.

▪ Streptococcus pneumoniae.

▪ Hemophilus influenzae.
❑ Z-N stain ( Ziehl Nielson Stain)
▪ For tuberculous meningitis.

❑ CSF Culture
▪ If appearance of bacteria on gram stained smears.

▪ Increased proteins or cell count.

▪ Sensitivity – 90%

▪ Before dispatch never keep the specimen in the refrigerator.

 ❖ Chemical examination
❑Total solids
1.Protein – 15 to 45 mg/dl
(albumin=50-70%)
(globulins=30-50%)
2. Glucose – 40-80 mg/dl
3. Chlorides – 700-750 mg/dl
4. Sodium – 144-154 meq/1
5. Potassium – 2.0-305 meq/1
6. Creatinine – 0.5-1.2 mg/dl
7. Cholesterol – 0.2-0.6 mg/dl
8. Urea – 6-16 g/dl
9. Uric acid – 0.5-4.5 mg/dl
 ❑ Globulin
▪ CSF under normal condition contain very little globin.

▪ Globulin is most important for detection of infection in CSF because

immunoglobulin develop within the CSF in case of infection.

▪ High level of globulin indicate:- Bacterial meningitis ,

malaria infection,
African trypanosomiasis,
paralysis
❖ Method - Pandy’s Method

❖Principle – Globulin are precipitated by a saturated solution

of phenol in water and cause a fine turbidity.

❖Reagent – Phenol - 10 g.
Distilled water - 150 ml
❖ Procedure:-
➢ Take 1 ml of pandy’s reagent in to a small test tube.

➢ Add 2-3 drop of CSF (specimen)

➢ Examine the tube after addition of each drop. Do not mix.

➢ Read the result immediately.

❖Result:- Depending upon the degree of precipitation of globulin .

trace, 1+ , 2+ , 3+ , 4+
SEMEN ANALYSIS
MLT Education Point
MLT Education Point
 WhatsApp Nu. - 9977002457

▪ Semen is a whitish or gray fluid secretion of the male

reproductive organ discharge from the urethra on ejaculation .

▪ It contain spermatozoa and seminal plasma.

▪ It is the collection of fluid from testes , seminal vesicles prostate

gland and bulbourethral gland.

❖ Volume of semen – 1 . 5 - 2ml/ejaculation.

❖ Reaction – It is alkaline in nature 7 . 5 PH.

❖ Colour – Whitish gray.

➢ Composition of semen WhatsApp Nu. - 9977002457

❑ Semen contains 5 % sperms and 95%of fluid part , which is

called seminal plasma .

❑ Seminal plasma contains the products from seminal vesicle and

prostate gland .

❑ It also has small amount of secretions from the mucus glands ,

particularly the bulbourethral gland.
 Semen MLT Education Point

5 % Sperm 95 % Seminal plasm

60 % Seminal vesicles 30% Prostate gland

5%
Acid phosphatase
Ascorbic acid Other gland
Bicarbonate
Fibrinogen
Clotting enzyme
Flavin
Cholesterol
Fructose Calcium
Inositol
Fibrinolysin
Pepsinogen Glucose
Citrate Lactate dehydrogenase
Citric acid Phospholipids
Phosphorylcholine Sodium, zinc
 MLT Education Point
➢ Semen Liquefaction
▪ At the time of ejaculation semen is liquid in nature.
▪ Coagulation and liquefaction occur in two stage process.
i. After ejaculation semen immediately coagulate by the action of
clotting enzyme (Fibrinogen) produced by seminal vesicles .
(Primary Clot)
ii. After some time it become liquid ones again by enzyme
produce in prostate gland.
 WhatsApp Nu. - 9977002457

(The semen liquefaction take place within 10 – 30 minutes).

When semen is ejaculated the sperm are nonmotile due to the viscosity
of coagulum when the coagulum dissolves the sperm become motile.
 MLT Education Point
➢ Sperm
▪ sperm / Spermatozoa are reproductive cell precent in normal semen .
Development of sperm in testis and largely stored in ampullary portion of
vas deferens then released in the process of ejaculation.

▪ Sperm constitute less than 5 % of the semen volume.

 MLT Education Point
❑ Head
▪ Length – 4 . 5um.

▪ Width – 3 um.

▪ Thick – 1 . 5 um.
Head consist of the nucleus and acrosome.

Nucleus – holds whole of chromatin.

Acrosome – Collection of many enzyme .[Beak down of across

membrane and enzyme released in the human FGT].
 WhatsApp Nu. - 9977002457
❑ Neck
Head is connected to the body by a short neck.

❑ Body
▪ Length – 5 . 9 um.

▪ Width – 1 um.

▪ The body of the sperm consists of a central core called axial

filament , cover by thin cytoplasmic capsule.
 MLT Education Point

❑ Tail
▪ Length – 50 um.

▪ It consist of 2 part mid piece / chief piece terminal segment.

[Tail means by which sperm moves abnormalities in tail cause of poor
sperm movement].
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WhatsApp Nu. - 9977002457
❑ Semen analysis in one of the first test due to evaluate a man’s fertility.

❑The test may also be used for after vasectomy.

❖ Specimen Collection
❑ Abstinence period 2 – 3 days.

❑ Production of specimen by masturbation.

❑ At least 3 sample of specimen collected.

 MLT Education Point
Examination of semen
Semen

Physical examination Microscopic examination Chemical examination

• PH
• Sperm count • Fructose
• Volume
• Color • Sperm motility
• Liquification • Sperm morphology
• Specific gravity
• Consistency /viscosity
 MLT Education Point
❑ Physical Examination
▪ Volume – 2 – 3 ml.

▪ Appearance – milky white or gray fluid

▪ if presence urine – urine contaminate semen in disturbance of bladder neck

function.

▪ Presence of blood in semen color pink called

hematospermia.

▪ pH of semen – Normal pH 7.5 - 8.0

 WhatsApp Nu. - 9977002457
❑ Liquefaction of semen – [Time –15 - 30 minute]

• In human semen is ejaculate in liquid form and after ejaculation if form a gel
like clot (by fibrinogen presence in seminal vesicle).

• After some time (15 – 30 minute) it become liquid by action of

prostatic enzyme proteases , pepsinogen , amylase, transaminase.

❑ Viscosity of semen– [Smooth and watery]

• Observed by – Taking the specimen in a Pasteur pipette

by drop by drop.
 MLT Education Point
❑ Microscopic Examination
• Collect a fresh sample of semen and wait for 15 – 30 minute for liquefaction
and examined as soon as possible.

Determination of morphology of sperm

▪ Procedure :–
 MLT Education Point
▪ Normal Observation :–
• Sperm Size – 50 – 70 um.

• Head Size – 3-6 um length.

• Head Cup – Light blue.

• Nuclear – Dark blue.

• Tail – About 90% of the total length.

 WhatsApp Nu. - 9977002457
▪ Abnormalities:–
• Abnormal head shaped.

• Size head.

• Double head.

• Middle section, absent ,swollen.

• Tail – double or absent , short , coiled.

 MLT Education Point

Head Defects Midpiece Defects Tail Defects

 MLT Education Point
▪ Determination of sperms motility :–

o Procedure
 WhatsApp Nu. - 9977002457

• Count the number of actively motile sperm out of the total count of 200.

• Calculate the percentage of sperm showing actual progressive motion.

• If 70 – 80 % are motile – normal (fertile).

 MLT Education Point

▪ Sperm movement may be graded from :–

• A (Fast progressive) – These are strongest and rapid progression .

• B (Non liner motility) – Slow progressive.

• C (Non progressive) – Sperm move their tail.

• D (Immotile) – Absence of forward progression.

 MLT Education Point
Grade – A Grade – B

Grade – C Grade – D
❑ Sperm Count WhatsApp Nu. - 9977002457

• After liquefaction gently mix the specimen.

• Requirement – Neubauer chamber WBC pipette.

• Reagent – Sodium bicarbonate (NaHCo3) – 5g

(semen diluent) 35% formation – 1ml
D/W – 99ml
 MLT Education Point
▪ Procedure :–
• Draw semen to the 0 .5 mark of a WBC pipette.

• Draw the semen diluting fluid to the 11 mark and mix well.

• Discard the few drop and charge the counting chamber and allow to
settle for about 5 min.

• Count sperm under high power in 4 corner squares of WBC count.

 MLT Education Point

▪ Calculation :–
No. of sperm 1ml of semen = N x 20 1000
4 x 0.1
= N x 50 , 000
 WhatsApp Nu. - 9977002457
❑ Chemical Examination
▪ Detection of fructose in semen :–

• Fructose is main sugar of semen.

• Absence of fructose result in completely immotile sperm.

• Normal Range – 150 – 300 mg/dl .

 MLT Education Point

• Method – Resorcinol

• Principle – When fructose is heated with resorcinol in an acid medium to

give red colored complex.

▪ Reagent –
Resorcinol – 50mg.
HCL – 33ml.
distilled water – 67ml.
 MLT Education Point
▪ Procedure –
• Take 1ml of the fructose reagent in test tube.

• Add 100 ul seminal fluid.

• Heat and boiling for 1 to 2 minutes and cooling slowly. Examine

the solution and report as follows.

• Negative – No change in color.

• Positive – Red – colored precipitate forms within 30 s.

 Abnormalities Definition
Aspermia Absence of semen
Azoospermia Absence of sperm in the semen
Teratozospermia Sperms that have morphological defects

Oligospermia Very low sperm count

Necrospermia All the sperms in the ejaculate are dead

Asthenozoospermia Poor sperm motility

Leucospermia A high level of white blood cells present in the semen

Hematospermia Appearance of blood in sperm
 MLT Education Point
Parameter Normal finding
Appearance Greyish ,white ,viscid, opaque
Viscosity normal viscosity can be poured drop by drop
Volume 1.5 to 5ml
Liquefaction time 20 – 30 minutes
pH 7 .2 to 7 . 8
Fructose Present 150 – 300 mg/dl
Sperm concentration /ml 15 -300 millions /ml
Sperm count Ejaculation 40 million
Total motility 40%
Viability 58%
Morphology 4%
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WhatsApp Nu. - 9977002457
STOOL EXAMINATION (मल परीक्षण)
➢ Stool is the waste matter discharged form bowels and
examination of stool (faces) sample to help diagnose
gastro intestinal disorders.

Including – Diarrhea , Dysentery, Constipation ,

parasitic infection , Peptic ulcer ,carcinoma ,
Malabsorption ,GIT bleeding ,(Bacterial infection) etc.
 MLT Education Point You Tube

➢ Collection of specimen (नमूने का संग्रह) →

➢ The Swab only for infants.
➢ Specimen collection in wide mouth ,clean , leak proof container
without contamination of urine

➢ Collected in the morning before breakfast.

➢ labelling the specimen.

➢ Collected at least 3 sample

Quantity-
5g- parasitological and bacteriologic.
50g for chemical analysis.
 MLT Education Point You Tube
Note
➢ Liquid stool examination within 30mint, 1hr after passing stool because
motile protozoa (amoebae and giardia) die quickly when they are
outside the body.

➢ For semi or soft formed specimen – 1hr.

➢ Preservation(संरक्षण) → WhatsApp Nu. - 9977002457

➢ If delay in specimen examination and for future

examination use of stool preservations.

➢ Use of preservatives :- maintain morphology of

protozoan parasites and prevent further development of
helminthic egg and larvae.

❖ Name of preservatives
➢ Cary – Blair medium
➢ Buffered glycerol saline
➢ Formalin solution
➢ Merthiolate iodine formalin (MIF)
➢ Polyvinyl alcohol
 MLT Education Point You Tube

Method of examination

Physical Microscopic Chemical

examination examination examination
• Color • Protozoans • Occult blood
• Consistency • Crystals • PH
• Amount • Fat globules
• Odor • Yeast
• Mucus • Bacteria
• Blood • Pus
 MLT Education Point You Tube
• Bulk – 100 – 200 gm/ day
• Color – Brown
• Water – up to 75%
• Ph – 6.0 – 7.5
• RBC – absent
• WBC – few
• E.C – present
• Crystal – calcium phosphate
Triple phosphate
• Fat – <7g / day
• Urobilinogen – 50 – 300 /24hr.
 Physical examination WhatsApp Nu. - 9977002457

➢ Consistency (गाढ़ापन) ⟶
Abnormal Expected Reasons
• Pale and bulky • poor fat digestion

• Hard , Dry • constipation.

Flattened and ribbon like • obstruction in the lumen of the bowel

narrowing colon.
• Watery • bacterial infection (diarrhea).

• Rice water stool • cholera

➢ Color (रं ग) ⟶
❖ Normal - Light to dark drown ( due to presence of bile pigment).
ABNORMAL POSSIBLE
COLOR REASONS
• Black • bleeding in upper GI tract
• Bright red • lower GI tract bleeding
• Fresh blood • anemic dysentery
mucus
• Clay colored • post hepatic jaundice( biliary
tract obstruction)
• (Greyish • abnormality in pancreas
white)
 MLT Education Point You Tube
➢ Blood (खून)→ Normal stool not contain blood.

o Abnormally
• Bacillary dysentery ,ulcerative colitis, intestinal
schistosomiasis and sever Trichuris infection.

• More blood in specimen but not visible called occult

blood.

➢ Mucus (pure or with blood) →

(बलगम रक्त के साथ)

Abnormally seen in-

• Constipation bacillary dysentery, ulcerative colitis
 MLT Education Point You Tube
Chemical examination
➢ PH → 𝟔. 𝟎 − 𝟕. 𝟓
➢ Normally – slightly acidic or slightly alkaline.

➢ Acidic (5) > - excessing carbohydrate diet.

➢ Alkaline (7.5)< - excess of protein in diet.

➢ Determination of PH :- (पीएच का ननर्ाारण)
WhatsApp Nu. - 9977002457

➢ Requirement – PH paper.

➢ Procedure –
• Dip PH paper in small quantity of the fecal material.

• Observe the color.

• Compare with color chart and record the PH

 MLT Education Point You Tube
➢ Determination of occult blood test
Occult blood are found in stool when any
hemorrhagic tendency in colon .

➢ Method – benzidine test.

➢ Requirement (मांग) –
• Glass slide
• Applicator stick.
• Hydrogen peroxide
• glacial acetic acid.
• Benzidine powder.

➢ Specimen (नमूना) – Feaces

➢ Procedure (प्रक्रिया) –
MLT Education Point You Tube

1. 4gm of benzidine mix in 100ml of glacial acetic acid.

2. A small amount of stool in mixed in 5 ml of distilled

water.

3. Take 1ml of mixed stool and 1ml of benzidine and glacial

acetic acid solution mixed well .

4. add few drop of H2O2 and observe the color formation.

5. Appearance of blue color indicate positive test.

WhatsApp Nu. - 9977002457
 MICROSCOPIC EXAMINATION
(सूक्ष्मदर्शी द्वारा परीक्षण)
❑ Microscopic examination of the stool in necessary to identify
helminths egg and larvae as well as protozoan cyst and trophozoites.

❑ Trophozoite :- Liquid and soft stool not in formed stool.

❑ Cyst :- Semi formed stool.

❑ Helminths egg :- Any type of specimen.

 MLT Education Point You Tube

MICROSCOPIC EXAMINATION

Wet mount Concentration

method
Saline wet Iodine wet
mount mount Flotation techniques Sediment techniques
• Saturated salt • Formalin ether
• Zinc sulphate Sedimentation
centrifuge
 WhatsApp Nu. - 9977002457
Wet mount

Saline wet Iodine wet

mount mount
 MLT Education Point You Tube
Saline wet mount
➢ U𝒔𝒆 𝒇𝒐𝒓 →
▪ Detection of trophozoite and cyst of protozoa egg and larva of
helminths Live motile trophozoites also detect .

➢ Requirement →
▪ Glass slide.
▪ Cover slip.
▪ Saturated saline solution.
▪ Normal saline.
▪ Small bottles.
 MLT Education Point You Tube
➢ Procedure →
▪ Place a drop of normal saline on a clean glass slide.

▪ Take a small quantity of faces by using applicator stick

and mix with a drop of normal saline.

▪ Palace a cover slip over if [Avoid air bubble formation

below Coverslip].

▪ Smear is then examined under microscope.

 Iodine wet mount WhatsApp Nu. - 9977002457

➢ Requirement →
▪ Same as saline wet mount.
▪ Lugol’s iodine solution.

➢ P𝐫𝐨𝐜𝐞𝐝𝐮𝐫𝐞 →
▪ Place a drop of Lugol’s iodine on the other
slide.
▪ Mix a small quantity of faces with a drop of
iodine solution.
▪ Place a cover slip over it and examine under
microscope.
 Faces MLT Education Point You Tube

Normal Iodine sample

saline solution

Mix the sample with

sodium chloride
on one side mix the
sample with iodine drop.

Place a cover slip

and labeled with
identification no.
 MLT Education Point You Tube
Concentration method

▪ Egg, cysts and trophozoites are often in such low number

in fecal material that they are difficult to be detected in
direct smear or mount there for concentration method
performed.

▪ Two commonly method used :-

1. Flotation techniques.
2. Sedimentation techniques.
 Concentration WhatsApp Nu. - 9977002457

Methods

Sedimentation Flotation
method method
▪ Saturated salt solution
▪ Modified formal
technique.
ether sedimentation
technique.
▪ Zink sulphate centrifugal
flotation technique.
 MLT Education Point You Tube
Flotation method
(तैरने की प्रणािी)
▪ Observed that all helminthic egg and protozoa cyst float
in saturated salt solution except unfertilized egg.

[1] Saturated salt solution technique

➢ Principle →
▪ The ova whose specific gravity is less than that of
the saturated saline will float on the top and are
collected by placing a glass slide on the surface.
 WhatsApp Nu. - 9977002457

½ saline Add small Fill full bottle Place Observe

solution amount with saline coverslip and under
specimen solution wait 15 – 30 microscope
(stool) mint
 MLT Education Point You Tube
➢ Procedure →

i. Fill half of the bottle with saturated saline solution.

ii. place small amount of fecal material in it by applicator stick.
iii. Mix well and fill the bottle completely with saturated saline.
▪ Place a cover slip on it.
▪ Leave for 15 – 30 mint.
▪ After 15 – 30 mint lift the slide gently.
▪ Place it on a glass slide and observe under microscope
(First under low power objective and later on
under high power objective of the microscopic).
 MLT Education Point You Tube
[2] Zinc sulphate centrifugal flotation technique
➢ Requirements →
▪ 33% zinc sulphate.
▪ Glass slides.
▪ Cover slips.
▪ Microscope.
▪ Centrifuge.
▪ Stool specimen.
 WhatsApp Nu. - 9977002457
➢ Procedure →
▪ Place about 1gm of faces in test tube.

▪ Add10.0ml of lukewarm D/W.

▪ Remove the coarse particles by straining

through a urine gauge.

▪ Collect the filtrate in a centrifuge tube and

centrifuge for one mint at 2,500 RPM.

▪ Pour off the supernatant and add about

5ml D/W to the sediments.
 MLT Education Point You Tube
▪ Shake well and centrifuge for one
minute at 2,500 RPM.

▪ Discard the supernatant and add 3ml

zinc sulphate solution.

▪ Mix well and add 2ml zinc sulphate solution.

▪ Mix well and centrifuge for about one mint

at 2,500 RPM.

▪ With a platinum wire loop sample is taken

from the surface on to a clean glass slide a
coverslip is put on and examined under
microscope.
 MLT Education Point You Tube
Sedimentation method
▪ Formal – ether sedimentation method.
➢ Requirements →
i. 10% formol – saline
ii. Ether.
iii. Centrifuge tubes.
iv. Glass slides.
v. Coverslips.
vi. Centrifuge.
vii. Microscope.
viii. Stool specimen (freshly collected).
 MLT Education Point You Tube
➢ Procedure →

▪ Mix about 1 g stool specimen in 7.0 ml

formol saline solution.
▪ Strain through a wire gauge and collect the
filtrate in a centrifuge tube.
▪ Add 3.0 ml ether in the filtrate and shake
vigorously for about one minute.

▪ Centrifuge at 2000 RPM for 2 minutes and

allow the contents to settle.

▪ Remove upper part of the fatty – debris

using a glass rod.
 MLT Education Point You Tube
▪ Remove supernatant leaving 1 to 2 drops.

▪ Place the mixed deposit on a glass slide

and place a coverslip on it.

▪ Examine under microscope.